The tick borne flaviviruses (TBFV) belong to the Family Flaviviridae, genus Flavivirus and comprise some of the most medically significant emerging and re-emerging viral pathogens. TBFV include tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Alkhurma hemorrhagic fever virus, deer tick virus (DTV) and Langat virus (LGTV). TBFV are transmitted to humans by ixodid ticks, and cause a spectrum of disease ranging from mild febrile illness to encephalitis, meningitis or hemorrhagic fevers. Other medically relevant flaviviruses include the mosquito-borne West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). Hence, research into the pathogenesis of TBFV will reveal insight into the biology of this globally important group of viruses. The research in our laboratory aims to identify and understand interactions between the TBFV and their hosts (both the arthropod and the mammal) critical to virus replication and pathogenesis. We study LGTV, a naturally attenuated member of the TBFV that shares approximately 80% identity with TBEV at the amino acid level. LGTV can be safely studied at Biosafety Level-2 (BSL-2) making it an excellent model to gain insight into the TBFV. Studies using LGTV will form the basis for work on the more virulent BSL-3 DTV and BSL-4 TBFV. Analysis of virus interactions with the invertebrate host. Ixodid ticks represent the natural reservoir of TBFV, are critical for virus persistence in nature, and are the major vector for infection of humans. Transmission of flaviviruses to humans occurs during tick feeding. We have developed a first generation microarray to investigate salivary gland transcriptional changes in Ixodes scapularis nymphs during feeding or after infection with LGTV. The immediate goal of this work is to identify tick salivary gland transcripts that play a role during feeding or for the replication or transmission of TBFV. The long-term goal of this work is to identify novel tick salivary gland genes that could be targeted for the development of vaccines that have the potential to disrupt tick feeding and/or flavivirus transmission. In 2012, we published the results of our microarray analysis of LGTV-infected and uninfected ticks has identified 39 differentially regulated salivary gland transcripts over a time course of feeding. Differentially regulated transcripts were annotated based on current knowledge from the Ixodes scapularis genome. These transcripts are classified as putative secreted proteins, lipocalins, Kunitz protease inhibitors, antimicrobial peptides, serpins, metalloproteases and transcripts of unknown function. . Pursuing these results in 2012, we expressed several recombinant tick salivary gland proteins. The proteins were expressed in a baculovirus system and are being tested for their effect on flavivirus replication in vitro on relevant cell types such as tick and mammalian cells. Also in 2012, we developed a novel in vivo model for flavivirus infection that mimics a tick bite by inoculating virus into the ear pinna. This route of inoculation was successful at inducing neuronal death, encephalitis and meningitis in 3 week old mice inoculated with Langat virus. In addition to studying viral pathogenesis in the mammalian host, this model allows for the analysis of the local skin infection in the ear and the draining lymph nodes. The model will enable us to assess in an in vivo system the importance of the recombinant salivary gland proteins on TBFV infection. These experiments will utilize deer tick virus, a North American TBFV in the TBEV serocomplex. Comparison of TBFV infection in mammalian and tick cells. A key difference between TBFV infection of vertebrate and arthropod host systems is that infection of ticks is persistent and non-cytolytic, whereas infection of mammalian hosts is typically acute and cytopathic. We are investigating the nature of this difference to identify responsible host and viral factors. In work submitted for publication, we have compared virus infection in mammalian and tick cell lines utilizing molecular virology as well as confocal microscopy, electron microscopy, and electron tomography. Flavivirus infection in mammalian cell lines is accompanied by massive proliferation and rearrangement of cellular membrane, derived mainly from endoplasmic reticulum. Electron tomography revealed virus-induced spherical vesicles thought to protect replicative intermediates from intracellular antiviral systems. In contrast to mammalian cells, TBFV-infection in tick cells shows delayed and decreased membrane proliferation. Additionally, electron tomography of infected tick cells shows a shift from spherical vesicles to tubular profiles, especially in the context of persistently infected cells. In the case of WNV and DEN, viral non-structural protein 4A has been implicated in the membrane rearrangements occurring during infection. Building upon our previous work, we are exploring the role of NS4A in TBFV infection and continuing to evaluate cellular mechanisms that allow persistent infection of tick cells. Viral determinants of pathogenicity. Previous data collected in this lab demonstrated that upon passaging of the tick-borne flavivirus, Langat virus (LGTV), in ISE-6 tick cells, three coding mutations (M:K115E, NS3:F604L, and NS4A:A81V) arose within the LGTV genome. This virus was subsequently demonstrated to have reduced neuroinvasiveness as examined by intraperitoneal infection of three week old laboratory mice. We obtained a LGTV infectious clone in order to identify which of these previously identified mutations were responsible for the altered neuroinvasiveness of this virus. The sequence of the LGTV infectious clone contained several coding mutations and thus these firstly were removed, exception of a D308A mutation within the E protein gene, which following several attempts, was not able to be removed. Following the removal of these mutations, the ISE-6 tick cell adaptations were introduced as single mutations or in combination to engineer a recombinant triple-mutant. These viruses were subsequently rescued in Vero cells, assessed by immunofocus assay, and demonstrated to display a small-plaque phenotype. Three week old mice were subsequently infected intraperitoneally with these viruses, as well as a non-recombinant wildtype LGTV, and examined for weight-loss and survival. Infection with the non-recombinant LGTV resulted in 70% survival whereas 100% of mice infected with the recombinant viruses survived until the end of the experiment (28dpi). The only coding difference between the non-recombinant wildtype LGTV and the wildtype recombinant LGTV is the E:D308A mutation. Further examination of published literature revealed that this mutation has been demonstrated to affect neuroinvasiveness and plaque size of another tick-borne flavivirus, Louping Ill virus. Thus it is therefore likely that this E:D308A mutation is the cause of the decreased neuroinvasiveness seen in our studies.